The biological effects of hsa-miR-1908 in human adipocytes

Lei Yang • Chun-mei Shi • Ling Chen • Ling-xia Pang •

Guang-feng Xu • Nan Gu • Li-jun Zhu • Xi-rong Guo •

Yu-hui Ni • Chen-bo Ji

Received: 18 November 2013 / Accepted: 10 November 2014 / Published online: 25 November 2014

� Springer Science+Business Media Dordrecht 2014

Abstract MicroRNAs (miRNAs) are small non-coding

RNAs involved in the regulation of gene expression. MiR-

1908 is a recently identified miRNA that is highly

expressed in human adipocytes. However, it is not known

what role of miR-1908 is involved in the regulation of

human adipocytes. In this study, we demonstrate that the

level of miR-1908 increases during the adipogenesis of

human multipotent adipose-derived stem (hMADS) cells

and human preadipocytes-visceral. Overexpression of miR-

1908 in hMADS cells inhibited adipogenic differentiation

and increased cell proliferation, suggesting that miR-1908

is involved in the regulation of adipocyte cell differentia-

tion and metabolism, and, thus, may have an effect on

human obesity.

Keywords Obesity � Hsa-miR-1908 � Human adipocytes �Adipogenisis

Introduction

The obesity pandemic has become a significant health

threat, and has resulted in considerably higher morbidity

and mortality rates and increasing healthcare costs [1]. In

humans, a pool of multipotent progenitor cells (such as

hMADS cells) persists in adipose tissue throughout life,

and is able to differentiate to form adipocytes [2–4].

Obesity is an excessive accumulation of adipose tissue, so

we can tell the importance of hMADS cells on human

obesity.

MicroRNAs (miRNAs) are endogenous, highly con-

served small non-coding RNA molecules of about 22

nucleotides in length that regulate gene expression by

binding to the 30 untranslated region (30-UTR) of thecomplementary target mRNA sequence, resulting in

translational repression and/or gene silencing [5–7].

Through their influence on target mRNAs, microRNAs are

involved in numerous metabolic processes, including as

tissue development, cell proliferation, and lipid metabolism

[8–10]. Since the discovery of the first miRNA, lin-4, in

Caenorhabditis elegans [11], thousands of miRNAs have

been experimentally or computationally identified in dif-

ferent species [12]. MiR-103, miR-143, miR-17–92, miR-

21, miR-30c, and miR-204/211 have been reported to

promote adipogenesis [13–18]; while miR-130 and the

miR-27 family inhibits adipogenesis [19, 20]. The func-

tions of miR-30c and miR-27b on human adipocytes dif-

ferentiation have been analyzed using hMADS cells; these

cells represent a novel and unique tool for the study of

human adipose cell development.

Hsa-miR-1908 was first discovered in human embryonic

stem cells in 2008 [21], but there was no functional study

of this miRNA until 2012 [22]. To date, the function of

miR-1908 in adipocytes is unknown. In this study, we

L. Yang � C. Shi � L. Chen � L. Pang � X. Guo � Y. Ni (&) �C. Ji (&)Institute of Pediatrics, Nanjing Medical University, No. 140

Hanzhong Road, Nanjing 210029, China

e-mail: niyuhui@hotmail.com

C. Ji

e-mail: chenboji@njmu.edu.cn

L. Yang � C. Shi � L. Chen � L. Pang � N. Gu � L. Zhu �X. Guo � Y. Ni � C. JiDepartment of Children Health Care, Nanjing Maternity and

Child Health Care Hospital Affiliated to Nanjing Medical

University, No. 123 Tianfei Road, Nanjing 210004, China

G. Xu

Department of Laboratory Medicine, The 82nd Hospital of the

People’s Liberation Army, No. 100 Jiankang East Road,

Huaian 223001, China

123

Mol Biol Rep (2015) 42:927–935

DOI 10.1007/s11033-014-3830-1

investigated the functional significance of miR-1908 in

hMADS cells.

Materials and methods

Cell culture and induction of differentiation

hMADS cells and HPA-v (Sciencell Research Laborato-

ries, San Diego, CA, USA) were both maintained in

preadipocyte medium (PAM; Sciencell Research Labora-

tories) supplemented with 5 % fetal bovine serum (FBS),

1 % preadipocyte growth supplement (PAGS) and 1 %

penicillin/streptomycin solution (P/S) at 37 �C in 5 % CO2.To induce differentiation, confluent hMADS cells and

HPA-v (day 0) were cultured in serum-free PAM con-

taining 5 lg/ml insulin, 1 lM dexamethasone, 0.5 mM3-isobutyl- 1-methylxanthine and 1 lM rosiglitazone. Themedium was changed every 2 days for the first 4 days.

Thereafter, the medium was replaced with serum-free PAM

containing 5 lg/ml insulin, which was changed every2 days until accumulation of lipid droplets was observed

(day 15).

Human tissue sample

The human adipose tissue (subcutaneous and visceral) of

obese (n = 16, BMI C 28) and matched normal (n = 12,

18.5 B BMI \ 24) as well as clinical data were obtainedfrom Nanjing Maternity and Child Health Care Hospital

Affiliated to Nanjing Medical University with the approval

of institutional review boards and the ethics committee of

Nanjing Medical University [23]. Informed consent was

obtained from all subjects.

Construction of a miR-1908 overexpressing lentiviral

vector: pGLV-H1-miR-1908-GFP/Puro

Using polymerase chain reaction (PCR), the miR-1908

minigene fragments were amplified from genomic DNA

prepared from hMADS cells, then cloned into a lentivirus

expression vector (pGLV-H1-GFP/Puro; GenePharma,

Shanghai, China) according to the manufacturer’s instruc-

tions. After sequencing to validate that the cloning had

occurred correctly, the expression plasmid and the pack-

aging plasmids were cotransfected into HEK-293T cells to

produce the pGLV-H1-miR-1908-GFP/Puro lentivirus

expression vector; the supernatant containing the lentiviral

vector particles was used to infect the hMADS cells. The

lentiviral vector with a non-targeting control was also

constructed in the same way.

Transfection of hMADS cells with the pGLV-H1-miR-

1908-GFP/Puro expression vector

hMADS cells were seeded onto 6 well plates (Corning,

New York, USA) at a density of 105 cells/well and 24 h

later were infected with the miR-1908 expression vector

and non-targeting control vector, using supernatant con-

taining lentiviral particles and 5 lg/ml hexadimethrinebromide (polybrene; Sigma, St. Louis, MO, USA),

according to the manufacturer’s instructions.

Oil red O staining of miR-1908 overexpressing hMADS

cells

Mature adipocytes (day 15) were rinsed three times in

phosphate-buffered saline (PBS), fixed in 4 % (m/v)

paraformaldehyde for 10 min, and then rinsed again with

PBS. The fat droplets in the cells were stained with 0.2 %

(m/v) oil red O (Sigma, St. Louis, MO, USA) in isopro-

panol for 30 min and then examined microscopically

(Olympus, Tokyo, Japan).

Quantification of triglyceride content

Mature adipocytes (day 15) were harvested in lysis buffer

and the cells homogenized. Triglyceride content was

measured using a tissue triglyceride assay kit (Applygen,

Beijing) using a DNM-9602 microplate reader (Perlong,

Beijing), according to the manufacturer’s instructions.

Quantitative real-time polymerase chain reaction

(q-RT-PCR) to determine miR-1908 expression levels

To assess miRNA expression levels, total RNA was isolated

from hMADS cells, HPA-v, adipocytes, the cells during the

adipogenesis and the human adipose tissue with TRIzol

(Invitrogen, Carlsbad, CA, USA) according to the manu-

facturer’s instructions, and reverse-transcribed into cDNA

using the TaqMan MicroRNA Reverse Transcription Kit

(Applied Biosystems, Foster City, CA, USA) in 15 ll RTreactions containing 200 ng of total RNA, 50 nM stem-loop

RT primer, RT buffer, 0.25 mM of each dNTP, 3.33 U/llMultiscribe reverse transcriptase (RT) and 0.25 U/ll RNaseinhibitor. Reactions conditions were as follows: 30 min at

16 �C, 30 min at 42 �C, and 5 min at 85 �C.For real-time PCR, 1 ll (1:20 dilution) of cDNA, 0.1 lM

TaqMan probe, 0.2 lM forward primer, 0.2 lM reverseprimer and TaqMan Universal PCR Master Mix II (Applied

Biosystems, Foster City, CA, USA) were included in 20 llreactions, using the following reaction conditions: 10 min at

95 �C then 40 cycles of 15 s at 95 �C and 1 min at 60 �C. All

928 Mol Biol Rep (2015) 42:927–935

123

real-time PCR experiments were carried out using the ABI

7500 real-time PCR system (Applied Biosystems, Foster

City, CA, USA). The RT primer, hsa-miR-1908 and miR-

let-7 PCR primers, and the TaqMan probe for hsa-miR-1908

and miR-let-7 were purchased from Applied Biosystems.

The real-time PCR results were analyzed and expressed

relative to the miRNA expression of CT (threshold cycle)

value. Small nucleolar RNA U6 (snRU6) and miR-103 are

both stably expressed in cells, so they were used as the

normalizing control [24, 25]. The miR-let-7 played an

important role in adipocyte differentiation and was highly

expressed in adipocytes [26].

Quantitative real-time polymerase chain reaction (q-RT-

PCR) to determine expression levels of PPARc(peroxisome proliferator-activated receptor c) and C/EBPa(CCAAT enhancer binding protein a) mRNA.

To assess the mRNA expression level of PPARc andC/EBPa, total RNA was reverse-transcribed into cDNAusing the High Capacity cDNA Reverse Transcription Kit

(Applied Biosystems, Foster City, CA, USA) in 20 ll RTreactions containing 480 ng of total RNA, 50 nM RT pri-

mer, RT buffer, 0.25 mM each dNTP, 5 U/ll MultiScribeRT and 2.5 U/ll RNase inhibitor. Reactions conditionswere as follows: 10 min at 25 �C, 120 min at 37 �C, and5 min at 85 �C.

Real-time PCR was performed as described above. The

PCR primer sets used were as follows: PPARc forwardprimer:

50-AAATATCAGTGTGAATTACAGCAAACC-30, reverseprimer: 50-GGAATCGCTTTCTGGGTCAA-30, probe:50FAM-TGCTGTTATGGGTGAAACTCTGGGAGATTCT-30TAMRA;C/EBPa forward primer: 50-TGTGCCTTGGAAATGCAAAC-30, reverse primer: 50-CGGGAAGGAGGCAGGAA-30, probe: 50 FAM-CACCGCTCCAATGCCTACTGAGTAGGG-30 TAMRA;18S forward primer: 50-CGCCGCTAGAGGTGAAATTC-30, reverse primer: 50-CATTCTTGGCAAATGCTTTCG-30, probe: 50 FAM-ACCGGCGCAAGACGGACCAGA-30 TAMRA. 18S is stably expressed incells, so it was used as the normalizing control.

Western blots to determine the protein expression levels

of PPARc and C/EBPa

Whole-cell lysates from hMADS cells were prepared using

radioimmunoprecipitation assay (RIPA) buffer (Beyotime,

Shanghai, China). The Whole-cell lysates were boiled in

5 9 Sodium Dodecyl Sulfonate (SDS) sample buffer

(Beyotime, Shanghai, China) for 5 min and subsequently

centrifuged at 12,000g for 15 min at 4 �C. The proteincontent was measured using a Pierce BCA Protein Assay

Kit (Thermo Scientific, New York, USA), according to the

manufacturer’s instructions. The extracted protein (30 lg)was separated on a 10 % sodium dodecyl sulfate poly-

acrylamide gel electrophoresis (SDS-PAGE) gel under

reducing conditions and then electroblotted onto nitrocel-

lulose membranes (Millipore, Bedford, MA, USA). After

blocking with 5 % skimmed milk, the membranes were

incubated overnight at 4 �C with the appropriate antibod-ies—PPARc antibody (Cell Signaling Technology, Dan-vers, MA, USA), C/EBPa antibody (Cell SignalingTechnology, Danvers, MA, USA) and anti-b-actin antibody(Abcam, Cambridge, MA, USA). Subsequently, the mem-

branes were hybridized with a secondary antibody conju-

gated with peroxidase (Santa Cruz Biotechnology, Dallas,

USA). The protein levels were quantitated using the

ChemiDoc system (Bio-Rad).

Cell proliferation assay

Cell growth was analyzed using a WST-8 Cell Counting

Kit-8 (CCK-8; Dojindo, Japan). Isolated cells were seeded

in a 96-well microplate at a density of 400 cells per well to

a final volume of 100 ll, and incubated for 4 h. Before anadditional 1 h incubation period at 37 �C, 10 ll of CCK-8was added to each well. Cell growth was measured at

OD450 nm using a DNM-9602 microplate reader (Perlong,

Beijing, China). This process was performed every 24 h

until the wells were full.

Analysis of apoptosis in miR-1908 overexpressing

hMADS cells

Cells were stained with Annexin V-FITC and propidium

iodide (PI) and analyzed for apoptosis according to the

manufacturer’s instructions (BD FACSCalibur, Mountain

View, CA, USA). Briefly, after being serum-deprived for

12 h, cells were cultured in PAM supplemented with 5 %

fetal bovine serum (FBS), 1 % preadipocyte growth sup-

plement (PAGS) and 1 % penicillin/streptomycin solution

(P/S) at 37 �C in 5 % CO2 again for 24 h. After that, cellswere trypsinized and washed with Dulbecco’s PBS, and then

resuspended in 400 ll of PBS. Subsequently, 100 ll of thiscell suspension was incubated in the dark with 5 ll of PI(50 lg/ml) and 10 ll Annexin V-FITC for 15 min at roomtemperature. Cells were then analyzed by flow cytometry.

Analysis of cell cycle distribution of miR-1908

overexpressing hMADS cells

The distribution of cells at specific cell cycle stages was

evaluated by assessment of DNA content using flow

Mol Biol Rep (2015) 42:927–935 929

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cytometry. To synchronize cell cycle, cells were serum-

deprived for 12 h, and then were cultured in PAM sup-

plemented with 5 % fetal bovine serum (FBS), 1 % prea-

dipocyte growth supplement (PAGS) and 1 % penicillin/

streptomycin solution (P/S) at 37 �C in 5 % CO2 again for24 h. After that, the cells were trypsinized and washed in

PBS, and fixed in 70 % ethanol at 4 �C overnight. Thencells were treated with RNase A (100 lg/ml) and PI(50 lg/ml) at 37 �C for 30 min. Subsequently, cells weresubjected to FACS analysis based on DNA content. Sam-

ples were analyzed using the FACStar cell sorter and

CellQUEST Pro (BD FACSCalibur, Mountain View, CA,

USA).

Statistical analyses

All transfection experiments were repeated six times. The

qRT-PCR assays were performed in triplicate and each

experiment was repeated at least three times. Data are

presented as the mean ± SD of three or more independent

experiments. Statistical analyses were performed using

one-way ANOVA or the paired Student’s t tests. The dif-

ferences between groups were considered to be statistically

significant when P \ 0.05.

Results

MiR-1908 is highly expressed in human adipocytes

To examine the expression of miR-1908 in hMADS cells,

HPA-v and adipocytes, we cultured the hMADS cells and

HPA-v, and induced both cell types to mature to adipo-

cytes. During the differentiation, we extracted total RNA at

day 0 and day 15. The expression of miR-1908 was

assessed by PCR; there was no difference in the expression

of miR-1908 in hMADS cells and HPA-v, and the relative

level of miR-1908 expression was greater in the differen-

tiation of day 15 (Fig. 1; P \ 0.05).

The expression of miR-1908 during the differentiation

of hMADS cells decreased first but increased later

As hMADS cells will eventually differentiate to become

mature adipocytes, we examined the level of miR-1908

expression using PCR during the differentiation process to

verify the effect of miR-1908. Using small nucleolar RNA

U6 and miR-103 as expression references, we demon-

strated that the level of miR-1908 expression was

decreased by day 4 of the differentiation process, but was

increased at day 15 (Fig. 2; P \ 0.05).

MiR-1908 expression is different in human adipose

tissue

We analyzed the expression of miR-1908 in human adipose

tissue (subcutaneous and visceral) of obese and matched

normal. The results revealed that the expression level of

miR-1908 of obese was lower than normal in human adi-

pose tissue-subcutaneous. On the other hand, the expres-

sion level of miR-1908 of obese was higher than normal in

human adipose tissue- visceral (Fig. 3).

We construct a miR-1908 overexpressing lentiviral

vector: pGLV-H1-miR-1908-GFP/Puro successfully

Lentiviral vectors, particularly those derived from human

immunodeficiency virus (HIV), are effective DNA delivery

systems [27]. To investigate the effects of miR-1908, we

constructed a lentiviral vector to overexpress miR-1908 in

hMADS cells. After the vector sequence was confirmed,

we introduced the pGLV-H1-miR-1908-GFP/Puro vector

into hMADS cells, and after 72 h, tested the level of miR-

1908 expression. In these cells, the level of miR-1908

expression was approximately fourfold higher than the

expression level in the negative control cells (Fig. 4a).

Additionally, we demonstrated that the expression level of

let-7 in the cells transfected with the miR-1908 overex-

pression vector was no different to the level of let-7

expression in the negative control cells (Fig. 4b), and

therefore we concluded that the overexpression of miR-

1908 has no effect on the expression of other endogenous

miRNAs.

Fig. 1 The miR-1908 relative expression in hMADS cells and HPA-v at the differentiation of day 0 and day 15 The expression levels of

miR-1908 in hMADS cells and HPA-v at the differentiation of day 0

were lower than the expression levels of miR-1908 in cells at the

differentiation of day 15. Data are representative of three independent

experiments; data are expressed as the mean ± SD. *P \ 0.05

930 Mol Biol Rep (2015) 42:927–935

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Overexpression of miR-1908 inhibits differentiation

of hMADS cells into adipocytes

To explore the effect of miR-1908 on adipocyte differenti-

ation, we used oil red O to investigate the effect of miR-

1908 on the forming of lipid droplets in the miR-1908

overexpression hMADS cells (Fig. 5a); we also measured

the triglyceride content of these cells (Fig. 5b). There were

no differences in number of lipid droplets or the triglyceride

content of transfected hMADS cells or negative control cell.

Overexpression of miR-1908 inhibits the expression

of PPARc and C/EBPa

The expression of PPARc and C/EBPa, which are key adi-pogenic differentiation transcription factors, was also exam-

ined. Using qRT-PCR to analyze mRNA expression, we found

that miR-1908 inhibited the transcriptional induction of

PPARc and C/EBPa at day 15 of differentiation (Fig. 5c).Using western blot analysis, the protein expression levels of

PPARc and C/EBPa were determined in human adipocytes ondays 4, 7, 10 and 15 of differentiation. On day 4, both PPARcand C/EBPa were expressed at a low level. At day 7, the levelsof both PPARc and C/EBPa were strongly increased. After15 days of differentiation, PPARc and C/EBPa expressionwas uncompletely blocked in the miR-1908-transfected cells

(Fig. 5d). These data suggest that miR-1908 inhibits adipocyte

differentiation by blocking the transcription of the adipogen-

esis determination genes PPARc and C/EBPa.

Overexpression of miR-1908 promotes the growth

of hMADS cells

The growth of adipose tissue is the result of adipocyte

hypertrophy and hyperplasia. We analyzed the proliferation

of the transfected hMADS cells in 144 h to evaluate the

effect of miR-1908 on adipocyte hyperplasia. The hMADS

cells transfected with miR-1908 grew faster than the neg-

ative control cells (Fig. 6). Therefore, miR-1908 promotes

the proliferation of hMADS cells.

Overexpression of miR-1908 does not influence

apoptosis of hMADS cells

As apoptosis is a process that is closely related to cell

proliferation, we examined the effect of miR-1908 on the

apoptosis of hMADS cells (Fig. 7a). The overexpression of

miR-1908 slightly inhibited the apoptosis of hMADS cells,

but this effect was not statistically significant (Fig. 7b).

Overexpression of miR-1908 induces S phase

during the cell cycle of hMADS cells

To further explore the function of miR-1908 in the pro-

motion of cell proliferation, we examined the distribution

Fig. 2 The miR-1908 relative expression during hMADS cellsdifferentiation The expression of miR-1908 decreased between day

0 to day 4 during hMADS cells differentiation, but by day 15 its

expression had increased. Data are representative of three

independent experiments; data are expressed as the mean ± SD

(The data were different from the above ones because they were from

different experiments). *P \ 0.05

Fig. 3 The miR-1908 relative expression in human adipose tissueThe expression level of miR-1908 in of obese was lower than normal

in human adipose tissue-subcutaneous. However, the expression level

of miR-1908 of obese was higher than normal in human adipose

tissue- visceral. Data are representative of three independent exper-

iments; data are expressed as the mean ± SD

Mol Biol Rep (2015) 42:927–935 931

123

of cells at different cell cycle phases in miR-1908 trans-

fected hMADS cells using flow cytometry (Fig. 8a). The

miR-1908 transfected cell population showed a substantial

increase of cells in S phase and a decrease in the number of

cells in G1 phase, compared with negative control cells

(Fig. 8b). This data suggests that miR-1908 promotion of

Fig. 5 The effect of miR-1908on hMADS cells differentiation.

a Lipid droplets stained with oilred O in transfected cells and

control cells (9100). There

were no differences in the

number of lipid droplets

between miR-1908-transfected

cells and the negative control

cells. b Triglycerideaccumulation was quantified

and normalized to protein

amount. There were no

differences between the

triglyceride content of miR-

1908-transfected cells and

negative control cells. b Theanalysis of PPARc and C/EBPamRNA expression using qRT-

PCR. The level of PPARc andC/EBPa mRNA expression inmiR-1908-transfected cells was

lower than in the negative

control cells. d Western blotanalysis of PPARc and C/EBPaprotein expression. MiR-1908

transfected cells had a low level

of PPARc and C/EBPa proteincompared with negative control

cells. Data are representative of

three independent experiments;

data are expressed as the

mean ± SD. *P \ 0.05

Fig. 4 The successful construction of the lentiviral miR-1908expression vector A lentiviral miR-1908 expression vector was

constructed and the level of miR-1908 expression was tested. a ThemiR-1908 expression level in transfected hMADS cells 72 h post-

transfection. The expression level of miR-1908 was higher than in the

negative control cells. b The expression level of let-7 in transfectedhMADS cells 72 h post-transfection. There was no difference

between the expression levels between the transfected cells and the

negative control cells. Data are representative of three independent

experiments; data are expressed as the mean ± SD. **P \ 0.01

932 Mol Biol Rep (2015) 42:927–935

123

hMADS cell growth by may be mediated through an

induction of S arrest.

Discussion

Obesity is an excessive accumulation of adipose tissue due

to energy imbalance, characterized by an increase in cell

number and volume, and is associated with health prob-

lems, including insulin resistance, type 2 diabetes, hyper-

tension, cardiovascular disease, and some cancers [28]. In

our previously research, we found that the expression of

many microRNAs in human adipocytes was higher than

that in human preadipocytes and hMADS cells. The other

members of our research group have studied the function of

several microRNAs [29, 30]. MiR-1908 is a recently

identified miRNA that is located in the first intron of

FADS1 on human chromosome 11 [21]. The functions of

its putative target genes were enriched in Wnt receptor

signaling pathways through beta-catenin, cell cycle, cell

proliferation and other biological processes. GnRH sig-

naling, MAPK signaling, insulin signaling, cell cycle signal

transduction pathways and pancreatic cancer were signifi-

cantly enriched. Both the MAPK signaling and the insulin

signaling pathways are known to be involved in adipocyte

differentiation and obesity [31, 32]. All of the signaling

pathways and biological processes that the putative target

genes are involved in are concerned with adipocyte dif-

ferentiation and metabolism, so we hypothesized that miR-

1908 would have an effect on the differentiation and

metabolism of human adipocytes. Additionally, miR-1908

has been identified as having a close relationship with the

processes of metastatic invasion, angiogenesis, and colo-

nization of melanomas [22]; it may be involved in the

malignant progression of chordoma [33]; and may partic-

ipate in the formation of hepatoma cells [34].

The process of adipogenesis can be divided into early,

intermediate and late stages. PPARc and C/EBPa are bothadipogenic transcription factors that are involved at the

early stage of adipocyte differentiation. Early stage adi-

pocytes have the potential to influence future adipocyte

biology, and influence the formation of adipose tissue;

consequently, the analysis of early adipogenic transcription

factors has been a primary target for anti-obesity strategies.

In this study, we identified that miR-1908 was upregu-

lated during differentiation of hMADS cells and HPA-v to

form adipocytes. Subsequent functional analyses demon-

strated that miR-1908 promotes the proliferation of

hMADS cells and inhibits their differentiation to adipo-

cytes, as evidenced by the downregulation of the key adi-

pogenic transcription factors PPARc and C/EBPa. Thisdownregulation ultimately resulted in decreased expression

of adipocyte marker genes (PPARc and C/EBPa) and an

Fig. 6 The effect of miR-1908 on hMADS cells proliferation. Weused CCK-8 to analyze the proliferation of transfected hMADS cells

and negative control hMADS cells. The miR-1908 transfected cells

grew faster than the negative control cells. Data are representative of

three independent experiments; data are expressed as the mean ± SD.

***P \ 0.001

Fig. 7 The effect of miR-1908 on hMADS cells apoptosis. a Apop-tosis in miR-1908 transfected hMADS cells was analyzed by flow

cytometry. b The overexpression of miR-1908 inhibited the apoptosisof hMADS cells, but this was not statistically significant. Data are

representative of three independent experiments; data are expressed

as the mean ± SD

Mol Biol Rep (2015) 42:927–935 933

123

increase in hMADS cell proliferation. Previously, it was

demonstrated that the antisense inhibition of specific

miRNAs (miR-10b, 15, 26a, 34c, 98, 99a, 101, 101b, 143,

152, 183, 185, 224, and let-7b) that are upregulated during

adipogenesis, did not affect adipocyte differentiation, as

determined by marker gene expression and the accumula-

tion of lipid droplets [35]. As there are many miRNAs and

genes that can regulate the adipocyte differentiation during

every stage, the specific mechanism of miR-1908 affecting

the adipocyte differentiation remains unknown and

demands further study.

The expression of miR-1908 differed between subcuta-

neous and visceral adipose tissue, suggesting that the

molecular mechanism of adipogenesis is site dependent [6].

Further study is needed.

Growth-arrested hMADS cells initiate clonal expansion

with adipogenic signals to re-enter the cell cycle progres-

sion. The cells become spherical, accumulate fat droplets

and are then converted into fully differentiated adipocytes

[36]. It has been suggested that miR-17-92 may modulate

adipocyte differentiation by regulating the re-entry and exit

of cells into the cell cycle [15]. In our study, miR-1908

inhibits adipocyte differentiation by promoting the prolif-

eration of hMADS cells and influencing the cell cycle

through expanding S phase and inhibiting G1 phase. How-

ever, how miR-1908 results in a rate decrease in G1 phase

and increase in S phase remains to be further researched.

In summary, we found that the miRNA miR-1908 is

highly expressed in human adipocytes. It inhibits the dif-

ferentiation of hMADS cells to form mature adipocytes and

promotes hMADS cell proliferation. Obesity is an exces-

sive accumulation of adipose tissue, so these findings

outline the importance of miR-1908 in human obesity.

Further experiments are required to elucidate the mecha-

nisms of miR-1908 in hMADS cells.

Acknowledgments This study was supported by grants from theNational Key Basic Research Program of China (2013CB530604),

Fig. 8 The effect of miR-1908 on the cell cycle in hMADS cells.a FACS assay of hMADS cells transfected with miRNA-control andmiR-1908 mimics. b Flow cytometry analysis. In miR-1908 trans-fected cells there are more cells in S phase and fewer cells in G1

phase, compared with the negative control cells. Data are represen-

tative of three independent experiments; data are expressed as

mean ± SD. *P \ 0.05; **P \ 0.01

934 Mol Biol Rep (2015) 42:927–935

123

National Natural Science Foundation of China (81100618), Natural

Science Foundation of Jiangsu Province China (BK2011107), Pro-

gram for Innovative Research Teams of Jiangsu Province (LJ201108)

and Nanjing Technological Development Program (201104013).

Conflict of interest No competing financial interests exist.

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The biological effects of hsa-miR-1908 in human adipocytesAbstractIntroductionMaterials and methodsCell culture and induction of differentiationHuman tissue sampleConstruction of a miR-1908 overexpressing lentiviral vector: pGLV-H1-miR-1908-GFP/PuroTransfection of hMADS cells with the pGLV-H1-miR-1908-GFP/Puro expression vectorOil red O staining of miR-1908 overexpressing hMADS cellsQuantification of triglyceride contentQuantitative real-time polymerase chain reaction (q-RT-PCR) to determine miR-1908 expression levelsQuantitative real-time polymerase chain reaction (q-RT-PCR) to determine expression levels of PPAR gamma (peroxisome proliferator-activated receptor gamma ) and C/EBP alpha (CCAAT enhancer binding protein alpha ) mRNA.Western blots to determine the protein expression levels of PPAR gamma and C/EBP alpha Cell proliferation assayAnalysis of apoptosis in miR-1908 overexpressing hMADS cellsAnalysis of cell cycle distribution of miR-1908 overexpressing hMADS cellsStatistical analyses

ResultsMiR-1908 is highly expressed in human adipocytesThe expression of miR-1908 during the differentiation of hMADS cells decreased first but increased laterMiR-1908 expression is different in human adipose tissueWe construct a miR-1908 overexpressing lentiviral vector: pGLV-H1-miR-1908-GFP/Puro successfullyOverexpression of miR-1908 inhibits differentiation of hMADS cells into adipocytesOverexpression of miR-1908 inhibits the expression of PPAR gamma and C/EBP alpha Overexpression of miR-1908 promotes the growth of hMADS cellsOverexpression of miR-1908 does not influence apoptosis of hMADS cellsOverexpression of miR-1908 induces S phase during the cell cycle of hMADS cells

DiscussionAcknowledgmentsReferences